Triglyceride (GPO)

(Liquid)
Reagent Set

Intended Use Do not use the reagent if:

For the in vitro quantitative determination of Triglycerides in serum or 1. The initial absorbance of the reagent is greater than 0.350 when measured
plasma. at 500nm against water in a cuvette with a one centimeter path length.
2. The reagent is turbid or displays evidence of bacterial contamination.
Clinical Significance
Triglycerides determinations are of interest in the diagnosis and treatment of Precautions
atherosclerosis, poorly controlled diabetes mellitus, nephrosis, liver disease, 1. This reagent set is intended for in vitro diagnostic use only.
or other diseases involving lipid metabolism. 2. The reagent contains sodium azide (0.01%) as a preservative. Do not
ingest. Avoid skin and eye contact. Sodium azide may react with copper or
Test Summary lead plumbing to form explosive metal azides. Upon disposal flush with large
amounts of water.
The triglycerides (GPO) method is based on the enzymatic determination of
3. All specimens and controls should be handled as potentially infectious. Use
glycerol using the enzyme glycerol phosphate oxidase (GPO) after hydrolysis
safe laboratory procedures. (NCCLS M29-T2)3
by lipoprotein lipase. The principle of this method was described by Fossati1
who coupled the reaction with the classical Trinder2 reaction sequence. This
single reagent procedure quantitates the total glycerides in serum including Specimen Collection and Storage
the mono and diglycerides, and the free glycerol fractions. This approach is 1. Fresh, clear, unhemolyzed serum is the specimen of choice. The specimen
the basis for this method. should be collected following the guidelines of NCCLS document H4-A3.4
2. The serum should be collected following a 12 hour fast, and separated from
Principle the clot as soon as possible. Avoid anticoagulants containing fluoride or
oxalate.
Lipase
3. Serum of plasma may be stored for one week at 2-8°C or for three months at
Triglycerides ------------------------------¾ Glycerol + Fatty Acids
–20°C.5
4. Frozen samples should be thawed at room temperature and mixed
GK
completely before analysis. Thawed samples should not be refrozen.
Glycerol + ATP ------------------------¾ Glycerol-1-phosphate + ADP

GPO Interferences
Glycerol - 1 - phosphate + O2------------------------¾DAP + H2O2 1. A number of drugs and substances affect the determination of
triglycerides.6,7 Young, et al8 have published a comprehensive list of these
POD substances.
H2O2 + 4-AA +4 -Chlorophenol ------------¾ Quinoneimine Dye + HCL + 2H2O 2. The method is not influenced by hemoglobin values up to 100mg/dl or by
bilirubin levels up to 12mg/dl (<5%).
Serum triglycerides are hydrolyzed to glycerol and free fatty acids by lipase. 3. Detergents can interfere with the action of lipase. Care should be taken to
In the presence of ATP and glycerol kinase (GK), the glycerol is converted to avoid contamination of laboratory equipment with detergents.
glycerol-1-phosphate. The glycerol-1-phosphate is then oxidized by glycerol
phosphate oxidase (GPO) to yield hydrogen peroxide. The condensation of Materials Provided
hydrogen peroxide with 4-chlorophenol and 4-aminophenazone (4-AA) in the Triglycerides (GPO) reagent
presence of peroxidase (POD) produces a red colored quinonimine dye
which absorbs at, or near 500nm. The intensity of the colored complex Materials Required but not Provided
formed is directly proportional to the triglycerides concentration of the 1. Accurate pipetting devices for delivering required sample and reagent
sample. volumes.
2. Test Tubes and racks
Reagent Composition 3. Timer
4-Chlorophenol 3.5mM, ATP >0.5mM, Magnesium salt 10 mM, 4- 4. Heating block or water bath (37°C)
Aminophenazone 0.3mM, Glycerol Kinase (microbial) >250 U/L, Glycerol 5. Spectrophotometer able to read at 500 nm
Phosphate Oxidase (microbial) >4500U/L, Peroxidase (horseradish) > 2000 6. Triglycerides standard or calibrator
U/L, Lipase (microbial) >200,000 U/L, buffer (pH 7.3 ± 0.1), surfactants,
stabilizers, and preservatives, including sodium azide (0.01%). Procedure (Automated-General)
Wavelength: 500nm
Reagent Preparation Assay Type: Endpoint
The reagent is ready to use. Sample/Reagent Ratio: 1:101
Reaction Direction: Increasing
Reagent Storage and Stability Temperature: 37°C
Store the reagent at 2-8°C. The reagent is stable until the expiration date Incubation Time: 300 sec.
appearing on the label when stored as directed. Protect from direct light. Low Normal: 44mg/dl
Avoid microbial contamination. High Normal: 148mg/dl

Phone: 734-487-8300 • Toll Free: 800-445-9853 • Fax: 734-483-1592 • www.pointescientific.com

 Triglyceride (GPO)
(Liquid)
Reagent Set

Instrument Application Procedures describing the use of this reagent on Expected Values
automated analyzers are available. Please contact our Technical Service 44-148 mg/dl (0.50-1.67 mmol/L)9
department for specific information.
Due to a wide range of conditions (dietary, geographical, age, etc.) believed to
Test Procedure (Manual) affect normal ranges, it is recommended that each laboratory establish its own
1. Label test tubes: “Blank”, “Calibrator/Standard”, “Patient”, “Control”, reference range.
etc.
2. Pipette 1.0 ml of reagent into the appropriate tubes and pre-warm to Performance
37°C.
1. Assay range: 0-1000mg/dl (0-11.3 mmol/L). Samples that exceed 1000
3. Add 0.010ml (10ul) of the appropriate sample to their respective tubes.
mg/dl should be diluted with an equal volume of saline and re-assayed.
Swirl gently to mix.
Multiply the result by two.
4. Incubate all tubes for five (5) minutes.
2. Comparison: A comparison was made between this method and a similar
5. After incubation, zero the spectrophotometer with “Blank” tube, at
GPO method using 167 samples ranging from 41 mg/dl to 1026 mg/dl. The
500nm. (500-520 nm is acceptable).
correlation coefficient was 0.999. Linear regression analysis gave the
6. Read and record the absorbance (Abs.) of all the tubes. The final color
following equation: This method y = 0.97 x – 4.5 Sy.x = 5.84.
is stable for at least 60 minutes.
3. Precision: Precision studies were performed following a modification of the
guidelines which are contained in NCCLS document EP5-T2.10
Limitations
The procedure is linear to 1000 mg/dl (11.3 mmol/L). Within Day Day to Day
Specimens above this limit must be diluted 1:1 with saline and reassayed. Mean S.D. C.V.% Mean S.D. C.V.%
Multiply the result by 2 to compensate for the dilution. 62.6 1.14 1.82 59.1 1.12 1.90
162.4 1.73 1.07 158.1 2.63 1.66
Calibration 301.9 3.24 1.07 299.3 3.65 1.22
Use an NIST-traceable Triglycerides standard or serum calibrator. The
procedure should be calibrated according to the instrument manufacturer’s 4. Sensitivity: The sensitivity for this product was investigated by reading the
calibration instructions. If control results are found to be out of range, the change in absorbance at 500nm for a saline sample, and serum samples
procedure should be re-calibrated. with known concentrations. Ten replicates were performed. The results of
this investigation indicated that, on the analyzer used, this product showed
Quality Control little or no drift on a zero sample. Under the reaction conditions described,
1mg/dl of triglycerides gives an absorbance of 0.001.
Standard practice for Quality Control should be applied to this procedure.
Commercially available controls (2 levels) should be used to monitor the
daily acceptable variations. Controls should be assayed at the beginning of References
each shift, whenever a new lot number of reagent is used, or following any 1. Fossati, P., Lorenzo, P., Clin. Chem. 28:2077 (1982).
instrument maintenance. A satisfactory level of performance is achieved 2. Trinder, P., Ann. Clin. Biol. Chem. 6:24 (1969).
when the analyte values obtained are within the “acceptable” range 3. NCCLS Document M29-T2, 2nd. Ed. (1991).
established by the laboratory. 4. NCCLS Document H4-A3, 3rd. Ed. (1991).
5. Tietz, N.W., Textbook of Clinical Chemistry, Philadelphia, PA, WB Saunders
Calculation Co. p888 (1986).
6. Martin, E., Hazards of Medication, Philadelphia, PA, J.B. Lippincott Co.
Triglycerides results are expressed as mg/dl or mmol/L.
pp.169-189 (1971).
7. Constantino, N.V., Kabat, H., Am. J. Hosp. Pharm. 30:24 (1973).
Triglycerides = Abs Unk x Conc. Std
8. Young, D.S., 3rd Ed. AACC Press, Washington DC (1990).
Abs Std
9. Rifkin, B.M., JAMA 250:1869 (1983).
Example:
10. NCCLS document “Evaluation of Precision Performance of Clinical
Abs Unk = 0.243
Chemistry Devices”, 2nd Ed. (1992).
Abs Std = 0.310
Conc. Std = 200 mg/dl
Manufactured by Pointe Scientific, Inc.
5449 Research Drive, Canton, MI 48188
Triglycerides = 0.243 x 200 mg/dl
0.310
European Authorized Representative:
Obelis s.a.
Triglycerides = 157 mg/dl
Boulevard Général Wahis 53
1030 Brussels, BELGIUM
Note: To convert the results into SI units (mmol/L), multiply the result (mg/dl)
Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: mail@obelis.net
by 0.0113.

Rev. 12/09 P803-T7532-01